The long term goal of the proposed studies is to understand the signal transduction mechanisms in the regulation of cell migration. Previous funding period focused on the role of focal adhesion kinase (FAK) and its downstream target Grb7 and a putative novel FAK inhibitor FIP200 in the regulation of cell migration. We identified and characterized Grb7 interaction with phosphoinositides through its PH domain and a role for Grb7 in mediating EphB1 stimulation of cell migration, showed the critical importance of focal contacts localization of the FAK signaling complexes, and characterized FIP200 inhibition of FAK activity and cell migration. We also found an interaction between FAK and N-WASP and showed that FAK phosphorylation of N-WASP regulates N-WASP subcellular localization and promotion of cell migration. In addition, we found an interaction between integrin p1 and 14-3-3p and studied the role of 14-3-3p in the regulation of cell spreading and migration, and an intra-molecular interaction between the N-terminal FERM-like domain and the kinase domain of FAK that regulate FAK activation in an auto-inhibitory mechanism. In preliminary studies, we identified an interaction between FAK and endophilin A2 and showed that endophilin A2 association with FAK and phosphorylation by FAK/Src complex regulated its interaction with dynamin and affected invasion of Src transformed cells. We are also in the process of creating mammary epithelial-specific FAK conditional knockout mice to study the role of FAK and its interaction with endophilin A2 in breast cancer metastasis in vivo. Lastly, to facilitate studies of directional cell migration on ECMgradients which better mimic in vivo conditions, we fabricated microfluidics devices to generate defined and quantitative FN gradients and showed directional cell migration on the gradients with novel properties. In this proposal, we will 1) determine the mechanism and role of FAK interaction with endophilin A2 in the regulation of cell migration and invasion, 2) investigate the potential role of FAK and its interaction with endophilin A2 in cancer metastasis in vivo, and 3) analyze the responses and roles of FAK and other signaling molecules in directional cell migration on defined FN gradients. These studies will enhance our understanding of the molecular mechanisms of signal transduction in cell migration and invasion which are critical factors in biological processes such as embryonic development, wound healing and cancer.